Inosine enhances the efficacy of immune‐checkpoint inhibitors in advanced solid tumors: A randomized, controlled, Phase 2 study

Abstract Background This study aimed to evaluate whether inosine enhances the efficacy of immune‐checkpoint inhibitors in human malignant solid tumors. Methods This single‐center, prospective, randomized, open‐label study was conducted, from January 2021 to December 2022, in Beijing Friendship Hospital, Capital Medical University, and participants were randomly assigned (1:1) to either the inosine (trial) or non‐inosine (control) group that received inosine (dosage: 0.2 g, three times/day) + PD‐1/PD‐L1 inhibitor or only PD‐1/PD‐L1 inhibitor ± targeted ± chemotherapy, respectively. Efficacy was assessed every 6 weeks (i.e., after every two–three treatment cycles). The primary endpoint was the objective response rate (ORR); the secondary endpoints were disease control rate, overall survival (OS), and progression‐free survival (PFS). The trial was registered at ClinicalTrials.gov (NCT05809336). Results Among the 172 participants with advanced malignant solid tumors, 86 each were assigned to the inosine and non‐inosine groups, wherein the median PFS (95% CI) was 7.00 (5.31–8.69) and 4.40 (3.10–5.70) months, respectively (hazard ratio [HR] 0.63; 95% CI 0.44–0.90, p = 0.011), and the ORR was 26.7% and 15.1%, respectively (p = 0.061). In the inosine and non‐inosine groups, the median OS was not reached and was 29.67 (95% CI 17.40–41.94) months, respectively (HR 1.05 [95% CI 0.59–1.84], p = 0.874). Compared with the non‐inosine group, the median PFS and ORR of the inosine group were significantly prolonged and improved in the multiple exploratory subgroup analyses. The safety analysis showed that Grades 3 and 4 adverse reactions occurred in 25 (29%) and 31 (36%) patients in the inosine and non‐inosine groups, respectively, and tended to decrease in the inosine group compared with the non‐inosine group. Conclusion Inosine had a tendency to enhance the efficacy of immune‐checkpoint inhibitors and reduced immunotherapy‐related adverse reactions.

Despite its clinical availability for decades, inosine has not been used for tumor immunotherapy.Considered with the preclinical evidence of its synergistic effect with ICI, the therapeutic potential of inosine deserves further evaluation.To further clarify whether inosine enhances the efficacy of immunotherapy, a prospective clinical study was conducted to compare the difference in efficacy between an inosine-treated group (trial group) and a non-inosine group (control group) for malignant advanced solid tumors.Inosine was used to enhance the efficacy of ICI or reverse ICI resistance and thus develop a new strategy for ICI combination therapy.

| Study design and participants
This single-center, prospective, randomized, open-label, Phase 2 study was conducted at the Department of Oncology, Beijing Friendship Hospital, Capital Medical University, Beijing, People's Republic of China, was approved by the Clinical Investigation Ethics Committee of Beijing Friendship Hospital, Capital Medical University (ethical approval number 2021-P2-152-02), and was conducted in accordance with the principles evinced in the Declaration of Helsinki.All participants provided written informed consent for data collection and study participation.

| Inclusion criteria
The inclusion criteria were as follows: (1) Histopathologically confirmed solid tumors that were locally advanced, recurrent, metastatic, or unresectable.

| Exclusion criteria
The exclusion criteria were as follows: (1) indication of active bleeding or perforation.(2) Systemic antitumor therapy with herbs or immunomodulatory drugs (including thymidine, interferon, and interleukin) within 2 weeks before the first dose of study treatment.(Appendix S3: detailed exclusion criteria).

| Assessments and follow-up
Antitumor efficacy was evaluated, according to RECIST v1.1, every 6 weeks, using computed tomography or magnetic resonance imaging and tumor markers.The treatment was administered until disease progression, death, or unacceptable toxic side effects; the investigator concluded that there was no benefit with continued treatment; other criteria for discontinuation were met (e.g., pregnancy, individual patient reasons, or concurrent illness); and the participant chose to withdraw from the study, withdrew informed consent, or was lost to follow-up.

| Sample size calculation
In this study, the unilateral β = 0.10 was considered (Power = 0.90; Alpha = 0.05); the samples of the two groups were equal, N1 (trial group) = N2 (control group), the predicted ORR of the combined group was 40%, and the ORR of the single group was 20%, and the number of cases in each group was calculated as 89, considering a dropout rate of approximately 5%, a total sample size of 188, with 94 participants in each group, was selected.The final specimen volume figures for the inquiry process were used.

| Outcomes
The primary endpoint was the ORR, the secondary endpoints were the disease control rate (DCR), overall survival (OS), and progression-free survival (PFS).Efficacy assessments were jointly completed after every two-three treatment cycles (every 6 weeks) and were undertaken by two experienced attending doctors in accordance with RECIST v1.1.The ORR represents the proportion of patients with the best overall effect evaluation of CR and PR, and the DCR denotes the proportion of patients with the best overall effect evaluation of CR, PR, and SD.The OS is the time from randomization grouping to death from any cause, PFS is the time from the start of randomization to the first documented disease progression or death from any cause.
Patients who received at least one treatment cycle were evaluated for safety.Adverse events were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 5.0.The occurrence of adverse events and immune-related adverse events throughout the study period and for up to 30 days (90 days for serious adverse events) after treatment discontinuation was monitored.Additionally, a drug-related assessment was performed to evaluate the safety in the inosine and non-inosine groups.

| Statistical analysis
Quantitative information for non-normal distributions was described using lower quartile, median, and upper quartile statistics, and qualitative information was described using absolute numbers and constitutive ratios.The Kaplan-Meier method was used to calculate the OS and PFS of the inosine and the non-inosine groups, and the log-rank test was used to ascertain the intergroup differences in OS and PFS.The chi-squared test was used to compare the differences in ORR, DCR, PFS, and OS rates for both groups.Receiver operating characteristic curves were employed to determine the optimal threshold for predicting the efficacy of inosine in subgroup exploratory analysis.The hazard ratio (HR) and 95% confidence interval (95% CI) were estimated using a Cox proportional hazards model.A two-sided p-value <.05 indicated statistical significance for the observed differences.Statistical analyses were performed using SPSS version 26.0.0 and R version 4.0.2.

| Participant characteristics
A total of 172 patients were enrolled from January 2021 to December 2022, with 86 patients each in the inosine and non-inosine groups.The cutoff date for data statistics was September 30, 2023.There was no significant intergroup difference in the baseline information of the inosine and non-inosine groups (Table 1).
Further exploratory subgroup analyses showed that the ORR significantly improved in the inosine group compared with that of the non-inosine group in the non-drinking, non-smoking, age >65 years, no adrenal metastasis, no bone metastasis, lung metastasis, or brain metastasis groups (Appendix S2: Table S1).

| Univariate and multivariate Cox regression analysis based on PFS and OS
Univariate Cox regression analysis was performed for PFS and OS in all patients and the inosine groups; subgroups with univariate Cox results of p < 0.100 were selected for multifactorial Cox regression analysis.The results of the univariate analysis showed that the number of lines, ENR, and inosine treatment were associated with PFS in all patients, whereas aging, brain metastasis, liver metastasis, bone metastasis, metastasis, age, ECOG, LDH, and NLR were associated with OS in all patients.
The multifactorial analysis revealed that the inosine treatment group exhibited reduced risk of disease progression compared to the non-inosine treatment group (HR 0.65, 95% CI 0.45-0.93,p = 0.019).Regarding OS, the  according to the five-point method in this trial, they were considered related to the study drug with relevance, very likely relevance, and possible relevance and unrelated to the study drug with possibly unrelated and unrelated.The Grades 3-4 adverse reactions in the inosine group included neutropenia in 8 (9%) cases, platelet decrease in 10 (12%) cases, alanine aminotransferase increased in 3 (3%) cases, aspartate aminotransferase increased in 4 (5%) cases.The Grades 3-4 adverse reactions in the non-inosine group included neutropenia in 11 (13%) patients, platelet decrease in 11 (13%) patients, alanine aminotransferase increased in 4 (5%) patients, and aspartate aminotransferase increase in five (6%) patients.No Grade 5 adverse reactions were observed in either group.All Grades 3 and 4 adverse reactions observed were stratified as follows: 25 (29%) and 31 (36%) in the inosine and non-inosine groups, respectively (p = 0.329; Table 3).

| DISCUSSION
Several clinical trials have demonstrated the effectiveness of ICI at 10-30% in all cancer treatment (CheckMate 078, 37 KEYNOTE-189, 38 KEYNOTE-024, 17,39 KEYNOTE-151, 40 KEYNOTE-181, 11 and KEYNOTE-177 41 ).4][35][36]42 Mager et al. administered a combination of inosine + CpG + anti-CTLA-4 in mouse models of colorectal, melanoma, and bladder cancer, all of which showed that inosine enhanced the antitumor immunity of anti-CTLA-4 in the presence of the co-stimulatory factor CPG. 26,43 In mice with B16 melanoma, Wang et al. showed that compared to treatment with a single PD-L1 inhibitor, an oral combination therapy regimen comprising inosine and PD-L1 inhibitor delayed tumor growth and prolonged survival.This combination significantly increased the number of CD8 + tumor-infiltrating T lymphocytes (TIL) expressing IFN-γ and TNF-α and resulted in a higher percentage of circulating CD8 + T cells expressing IFN-γ and TNF-α in the draining lymph nodes and the spleen. 36nosine supplementation enhanced the efficacy and durability of T cell-based cancer immunotherapy in a preclinical mouse model. 36Zhang et al. found that inosine enhanced the immunotherapeutic response, both in mice and humans, by acting on UBA6 of tumor cells, increasing the immunogenicity of tumor cells, and thus enhancing the killing effect of T cells. 35netheless, only preclinical, but not clinical, reports have shown that the microbial metabolite inosine can enhance the therapeutic efficacy of ICI.In this study, the mPFS in the inosine group was prolonged by 2.60 months compared with the non-inosine group, and inosine reduced the risk of disease progression by 37%, which was statistically significant.The mOS did not differ significantly; however, this was possibly related to several factors, including the subsequent treatment regimen and individual circumstances.The ORR in the inosine group was approximately 10% higher than in the non-inosine group, and thus lacked statistical significance.Both mPFS and ORR were significantly prolonged and improved in multiple exploratory subgroup analyses.This clinical trial showed that inosine had a tendency to enhance the efficacy of ICIs and delay disease progression, and these findings are consistent with the results of previous animal and preclinical studies.
In terms of safety, Grades 3 and 4 adverse reactions were observed in 25 (29%) and 31 (36%) patients in the inosine and non-inosine groups, respectively, and inosine likely reduced immunotherapy-related adverse reactions.The results of this trial suggest that, compared with that in the non-inosine group, the mPFS was significantly prolonged in the inosine group.Inosine combined with PD-1/PD-L1 inhibitors has a synergistic effect that will provide a new option for overcoming immunotherapy resistance.Inosine tablets have been used clinically as a long-term adjuvant treatment for hepatitis, is safe and economical, and will not impose a huge economic burden on the patient.In the subgroup analysis, the median PFS for lung cancer and digestive system malignancies tended to increase in the inosine group.Future Phase III clinical trials should focus on the selection of a single tumor species of lung or gastrointestinal system cancer in an expanded sample size for the verification of these findings.OS is related to the patients' general status before enrollment, age, disease stage, number of previous treatment lines, follow-up treatment opportunities, and the follow-up treatment protocols after disease progression.Thus, future phase III clinical studies should balance these factors and provide a detailed assortment of these factors for subgroup analysis to more accurately determine the relationship between drugs and OS.Owing to the limited sample size of this trial, the single-center design, enrolled multi-kinds of cancers, and non-pre-designed subgroups.Therefore, the results of the trial have some limitations and require further validation in a multicenter clinical trial with a larger sample.

| CONCLUSION
In this trial, the median PFS in the inosine group was 2.6 months longer than that in the non-inosine group and the risk of disease progression was reduced by 37%.The median OS was not reached in the inosine group and was 29.67 months in the non-inosine group.The ORR of the inosine group was higher than that of the non-inosine group, although the difference was not statistically significant.Inosine had a tendency to enhance the efficacy of ICIs and reduced immunotherapy-related adverse reactions in clinical applications.

F I G U R E 1
Kaplan-Meier method for estimating progression-free survival (PFS) and overall survival (OS) for the inosine and noninosine groups.(A) PFS in the total study population.(B) OS in the total study population.(C) PFS in the cancer group.(D) OS in the lung cancer group.(E) PFS results in gastrointestinal malignancies.(F) OS results in gastrointestinal malignancies.HR, hazard ratio, 95% CI, 95% confidence interval.mortality risk increased in the brain metastasis group compared with the no brain metastasis (HR 3.41, 95% CI, 1.59-7.30,p = 0.002), in the hepatic metastasis group compared with the no hepatic metastasis group (HR 2.40, 95% CI 1.29-4.47,p = 0.006), in the age >65 years group compared with age ≤65 years group (HR 1.93, 95% CI

F I G U R E 4
Univariate and multivariate Cox regression analysis of the inosine group, with regard to the PFS and OS.OS, overall survival; PFS, progression-free survival.| 11 of 14 ZHAO et al.
Intergroup comparison of the baseline information of the participants.
Intergroup comparison of efficacy indicators.
All adverse reactions in both the study groups.
T A B L E 3